Think positive: Proposal of a simple method to create reference materials in the frame of microplastics research.

In the context of the harmonization of methodologies employed to isolate and count microplastics in samples or to organize ring trials tests, the use of reference materials, i.e. samples with controlled amount of particles is required. The method proposed here uses transparent, sealed capsules containing in-house generated microplastics as a convenient way to generate microplastic reference materials. This method is a simple approach for adding particles to samples without risk of loss during particle extraction or transporting/handling.•Low-cost and easy-to-use preparation of heterogeneous mix of microplastic reference particles•Possibility to control microplastic size, shape, and polymeric composition•Applicable to many protocols and wide range of applications on water, sediments and biota.

Oral exposure to polyethylene microplastics alters gut morphology, immune response, and microbiota composition in mice.

The ubiquitous and growing presence of microplastics (MPs) in all compartments of the environment raises concerns about their possible harmful effects on human health. Human exposure to MPs occurs largely through ingestion. Polyethylene (PE) is widely employed for reusable bags and food packaging and found to be present in drinking water and food. It is also one of the major polymers detected in human stool. The aim of this study was to characterize the effects of intestinal exposure to PE MPs on gut homeostasis. Mice were orally exposed for 6 weeks to PE microbeads of 2 different sizes, 36 and 116 µm, that correspond to those found in human stool. They were administrated either individually or as a mixture at a dose of 100 µg/g of food. Both PE microbead sizes were detected in mouse stool. Different parameters related to major intestinal functions were compared between control mice, mice exposed to each type of microbead, or co-exposed to the 2 types of microbeads. Intestinal disturbances were observed after individual exposure to each size of PE microbead, and the most marked deleterious effects were found in co-exposed mice. At the histomorphological level, crypt depth was increased throughout the intestinal tissues. Significant variations of gene expression related to epithelial, permeability, and inflammatory biomarkers were quantified. Defective recruitment of some intestinal immune cells was observed from the proximal portion of the small intestine to the colon. Several bacterial taxa at the order level were found to be affected by exposure to the MPs by metagenomic analysis of cecal microbiota. These results show that ingestion of PE microbeads induces significant alterations of crucial intestinal markers in mice and underscores the need to further study the health impact of MP exposure in humans.

An Irgafos® 168 story: When the ubiquity of an additive prevents studying its leaching from plastics.

Plastic pollution is a source of chemical to the environment and wildlife. Despite the ubiquity of plastic pollution and thus plastic additive in the environment, plastic additives have been studied to a limited extend. As a prerequisite to a study aiming to evaluate the leaching of a common additive used as an antioxidant (Irgafos® 168) from polyethylene microparticles, an inventory of the potential background contamination of the laboratory workplace was done. In this study, Irgafos® 168 (tris(2,4-ditert-butylphenyl) phosphite) and its oxidized form (tris (2,4-ditert-butylphenyl) phosphate) were quantified in different laboratory reagents, including the plastic packaging and the powders, using Pyrolysis-GC/MS. At least one form of Irgafos® 168 was detected in all tested laboratory reagents with higher concentrations in caps and bottles as compared to the powders. Additionally, oxidized Irgafos® 168 was also found in the reverse osmosed and deionized water container used in the laboratory. The same profile of contamination, i.e. higher concentration of the oxidized form and higher concentrations in acidic reagents, was observed when comparing the reagent and their respective containers suggesting that the additive is leaching from the container into the powder. Overall, this study demonstrates that the antioxidant additive Irgafos® 168 is ubiquitous in the laboratory workplace. Plastic additives such as Irgafos® 168 can therefore largely interfere and biased ecotoxicological and toxicological studies especially using environmentally relevant concentrations of microplastics. The source, fate and effects of plastic additive from plastic debris should be carefully considered in future studies that require setting up methods to overcome these contaminations.

Occurrence and identification of microplastics in beach sediments from the Hauts-de-France region.

The present work was carried out to quantify microplastics (MP) from three sandy beaches along the Côte d'Opale coastline located in the Hauts-de-France region of northern France. Three different study sites located along the English Channel were investigated due to different levels of anthropopression and hydrodynamic conditions. Sediments were collected at three different tide lines: high tide line (HTL), middle of the intertidal zone (IZ), and low tide line (LTL), to investigate the effects of tide line on microplastic contamination. Particles and fibers were counted and colors were recorded; polymer identification was then performed using pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS). Particle and fiber abundances ranged from 23.4 ± 18.9 to 69.3 ± 30.6 items kg-1 dry weight sediment, with a trend towards fiber predominance, were observed. No difference in particle and fiber abundance was found between the different beaches and tide lines, except for Boulogne-sur-Mer, where the particle number was significantly different between tide lines. Major polymers identified were polyethylene (36.6%) and polypropylene (10.7%). This citizen science project provided preliminary data about the abundance and polymeric nature of MP along the Côte d'Opale coastline.

Microplastic contamination and pollutant levels in mussels and cockles collected along the channel coasts.

Nowadays, environmental pollution by microplastics (<5 mm; MP) is a major issue. MP are contaminating marine organisms consumed by humans. This work studied MP contamination in two bivalve species of commercial interest: blue mussel (Mytilus edulis) and common cockle (Cerastoderma edule) sampled on the Channel coastlines (France). In parallel, 13 plastic additives and 27 hydrophobic organic compounds (HOC) were quantified in bivalves flesh using SBSE-TD-GS-MS/MS to explore a possible relationship between their concentrations and MP contamination levels. MP were extracted using a 10% potassium hydroxide digestion method then identified by µ-Raman spectroscopy. The proportion of contaminated bivalves by MP ranged from 34 to 58%. Blue mussels and common cockles exhibited 0.76 ±â€¯0.40 and 2.46 ±â€¯1.16 MP/individual and between 0.15 ±â€¯0.06 and 0.74 ±â€¯0.35 MP/g of tissue wet weight. Some HOC and plastic additives were detected in bivalves. However, no significant Pearson or Spearman correlation was found between MP loads and plastic additives or HOC concentrations in bivalve tissues for the two species.

Validation of standard method EN ISO 19343 for the detection and quantification of histamine in fish and fishery products using high-performance liquid chromatography.

In contaminated fish, bacterial decarboxylases produce histamine from histidine, thereby causing scombroid fish poisoning. European Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs requires using a fully validated, standardized reference HPLC method for detecting and quantifying histamine. After optimizing this reference method for the quantification of histamine in fish muscle, we organized an inter-laboratory study in 2013 across nine laboratories from seven European countries using defined criteria of method performance. The optimized, validated method was standardized (Standard EN ISO19343) as part of Mandate M381 from the European Commission to the European Committee for Standardization (CEN), signed in December 2010. The standard method was validated for three types of foodstuffs (fish with enzymatic maturation, fish without enzymatic maturation and fish sauce).

Optimization, performance, and application of a pyrolysis-GC/MS method for the identification of microplastics.

Plastics are found to be major debris composing marine litter; microplastics (MP, < 5 mm) are found in all marine compartments. The amount of MPs tends to increase with decreasing size leading to a potential misidentification when only visual identification is performed. These last years, pyrolysis coupled with gas chromatography/mass spectrometry (Py-GC/MS) has been used to get information on the composition of polymers with some applications on MP identification. The purpose of this work was to optimize and then validate a Py-GC/MS method, determine limit of detection (LOD) for eight common polymers, and apply this method on environmental MP. Optimization on multiple GC parameters was carried out using polyethylene (PE) and polystyrene (PS) microspheres. The optimized Py-GC/MS method require a pyrolysis temperature of 700 °C, a split ratio of 5 and 300 °C as injector temperature. Performance assessment was accomplished by performing repeatability and intermediate precision tests and calculating limit of detection (LOD) for common polymers. LODs were all below 1 µg. For performance assessment, identification remains accurate despite a decrease in signal over time. A comparison between identifications performed with Raman micro spectroscopy and with Py-GC/MS was assessed. Finally, the optimized method was applied to environmental samples, including plastics isolated from sea water surface, beach sediments, and organisms collected in the marine environment. The present method is complementary to µ-Raman spectroscopy as Py-GC/MS identified pigment containing particles as plastic. Moreover, some fibers and all particles from sediment and sea surface were identified as plastic. Graphical abstract ᅟ.

Microplastics in seafood: Benchmark protocol for their extraction and characterization.

Pollution of the oceans by microplastics (<5 mm) represents a major environmental problem. To date, a limited number of studies have investigated the level of contamination of marine organisms collected in situ. For extraction and characterization of microplastics in biological samples, the crucial step is the identification of solvent(s) or chemical(s) that efficiently dissolve organic matter without degrading plastic polymers for their identification in a time and cost effective way. Most published papers, as well as OSPAR recommendations for the development of a common monitoring protocol for plastic particles in fish and shellfish at the European level, use protocols containing nitric acid to digest the biological tissues, despite reports of polyamide degradation with this chemical. In the present study, six existing approaches were tested and their effects were compared on up to 15 different plastic polymers, as well as their efficiency in digesting biological matrices. Plastic integrity was evaluated through microscopic inspection, weighing, pyrolysis coupled with gas chromatography and mass spectrometry, and Raman spectrometry before and after digestion. Tissues from mussels, crabs and fish were digested before being filtered on glass fibre filters. Digestion efficiency was evaluated through microscopical inspection of the filters and determination of the relative removal of organic matter content after digestion. Five out of the six tested protocols led to significant degradation of plastic particles and/or insufficient tissue digestion. The protocol using a KOH 10% solution and incubation at 60 °C during a 24 h period led to an efficient digestion of biological tissues with no significant degradation on all tested polymers, except for cellulose acetate. This protocol appeared to be the best compromise for extraction and later identification of microplastics in biological samples and should be implemented in further monitoring studies to ensure relevance and comparison of environmental and seafood product quality studies.

Occurrence of the three major Vibrio species pathogenic for human in seafood products consumed in France using real-time PCR.

Vibrio spp. have emerged as a serious threat to human health worldwide. Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus are of particular concern as they have been linked to gastrointestinal infections and septicemia associated with the consumption of raw or undercooked seafood. We developed hydrolysis probe-based real-time PCR systems with an internal amplification control for the detection of these species. We applied these systems to a total of 167 fresh or frozen crustacean, fish and shellfish samples consumed in France. Of them, 34.7% (n=58) were positive for Vibrio. V. parahaemolyticus was the most common, in 31.1% of samples, followed by V. vulnificus in 12.6% and V. cholerae in 0.6%. Furthermore, V. parahaemolyticus and V. vulnificus were present simultaneously in 9.6% of samples. Virulence genes (tdh and trh sequences) were present in 25% of the V. parahaemolyticus-positive samples. The V. cholerae strain detected was non toxigenic. The densities of V. parahaemolyticus and V. cholerae ranged from <10(2) to 10(4)bacteria/g of seafood. All samples positive for V. vulnificus displayed low-level contamination with fewer than 10(2)bacteria/g. Our findings indicate that seafood consumption presents a potential risk to human health in France and highlight the importance of tools for a preventive consumer protection policy.

Evolution of volatile compounds and biogenic amines throughout the shelf life of marinated and salted anchovies (Engraulis encrasicolus).

Producers of processed anchovies have developed hazard analysis and critical control points (HACCP) to guarantee the quality of their products. Nonetheless there is a lack of objective data to determine products' shelf life. The quality of a product is usually established on the basis of its safety and organoleptic properties. These parameters were assessed by monitoring the profiles of volatile compounds and quantitating six biogenic amines in samples of two types of processed anchovies during their shelf life. With regard to biogenic amines, quantities were below the regulatory limits throughout shelf life, except when a temperature abuse was applied for marinated samples. Moreover, this work highlights an optimum volatile profile at 5 and 6 months of storage for salted and marinated anchovies, respectively. This is the result of a higher content of six aldehyde and nine ketone compounds, mainly from lipid oxidation.